The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts.

We compared two protocols for the expansion of human mesenchymal stromal cells (hMSCs) starting from diagnostic samples of BM aspirates (2-5 ml) or using the remnants in the bag and filter at the end of the BM infusions. The protocols differed in the presence of either 10% fetal bovine serum (FBS) or 5% platelet lysate (PL). We obtained a significantly (P=0.02) better expansion with PL, obtaining a median 1010-fold compared to 198-fold with a selected batch of FBS and in fewer days (29.8 in PL versus 41.4 in FBS). Overall, we recovered a variable number from 54.8 x 10(6) to 365 x 10(6) hMSCs in PL versus a variable number from 2.7 x 10(6) to 31 x 10(6) in FBS. No difference could be found in terms of gross morphology, differentiation potential, surface markers and immunological properties (inhibition of allogeneic PHA response and mixed lymphocyte reaction) of cells expanded with PL or FBS. The preparations were found within the range of acceptability for all the quality control criteria. Due to the clinical grade nature of the PL and the reproducibility of separate preparations, we propose this method to obtain hMSCs even from minute amounts of BM cells.

Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.

We have used a standardized 21-day expansion protocol to produce cytokine-induced killer (CIK) cells starting from very small amounts of nucleated cells (approximately 15 x 10(6) cells) isolated from cord blood. Mononuclear cells are stimulated with anti CD3 (OKT3) and IFNgamma and then expanded with IL-2. Moreover, we show that washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500 x 10(6) CIK by the same expansion protocol. CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels. The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions and therefore these observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.

The human A-myb protein is a strong activator of transcription.

The A-myb gene belongs to the family of the c-myb proto-oncogene. We report here the cloning from a B lymphocyte cDNA library of the previously missing 3' half of the human A-myb cDNA, thus closing the previously still incomplete open reading frame. Analysis of the homologies between the different myb proteins reveals four domains of high conservation. We show, using a polyclonal rabbit antibody, that the 90 kd human A-myb protein is nuclear and that it activates transcription from the KHK-CAT reporter 6-10 times more strongly than c-myb in NIH3T3 cells. The transactivating function of A-myb depends on the presence of the myb binding site in the reporter, and on both the DNA binding and acidic domains of the A-myb protein. The bacterially expressed protein protects the myb binding sites of the reporter in footprint experiments. Binding of the A-myb protein is shown in gel retardation assays to be specific for the classical c-myb recognition sequence PyAACG/TG. In addition, like c-myb, A-myb binds more strongly to the MIM-A synthetic oligonucleotide that carries the TAACGG sequence than to the MBS-I oligonucleotide containing TAAGTG. Finally, DNA binding activity is demonstrated to require the N-terminal portion of the protein containing the three tandem repeats of amino acids conserved in all myb proteins. We have thus shown that the A-myb protein is a strong activator of transcription and that this activity depends on both the DNA-binding and acidic domains.

Isolation and characterization of the human A-myb promoter: regulation by NF-Y and Sp1.

The A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound effects on the growth and/or differentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the first exon, part of the first intron as well as upstream regulatory sequences. Multiple transcription start sites have been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (-183-14), of a positive upstream (-538-183) and a negative downstream regulatory region (NRE) (+83+374) have been defined. The NRE is promoter- and orientation-independent but position specific. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Spl and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction.

How can oncogenic transcription factors cause cancer: a critical review of the myb story.

Many transcription factors have been found to be oncogenic either when functionally altered through fusion with other proteins or through deregulated expression. However, still little is understood about the mechanism by which these proteins can transform cells of different origin. The Myb transcription factor is emblematic in this respect. Several forms of Myb can transform hematopoietic cells of different lineages in the chicken and Myb has been shown to be required for murine fetal hematopoiesis and to regulate the growth and differentiation of hematopoietic cells of several animal species. The role of the transcriptional activity of Myb and its possible gene targets in transformation are critically discussed.

Clinical grade expansion of MSCs.

Producing advanced therapy medicinal products (ATMP) according to Good Manufacturing Practice (GMP) guidelines represents a global challenge for the expansion of cells intended for human use. Mesenchymal stromal cells (MSCs) from different sources are one of the most actively developed cell type for a variety of clinical applications in cellular therapy. Complying with GMP means defining accurately both the production process and the release criteria required for a final safe product. We have here reported our manufacturing experience on 103 consecutive clinical-grade in vitro expansions of both bone marrow-derived and umbilical cord-derived mesenchymal stromal cells together with description of methods and reagents utilized in our Cell Factory. The same animal- and serum-free medium, additioned with human platelet lysate, has been used for all the expansions performed. This is the largest experience published so far with this alternative and clinical-grade reagent (compared to the traditional fetal bovine serum) and shows the feasibility and the reproducibility of the method. Indeed, we have been able to produce a sufficient number of MSCs to treat 57 patients so far, enrolled in 7 different experimental phase I/II protocols.

Characterization of CD20-transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of graft-versus-host disease.

We have previously proposed the CD20 molecule as a novel suicide gene for T lymphocytes in the context of allogeneic bone marrow transplantation, because CD20 can be used both as a selection marker and as a killer gene after exposure to the anti-CD20 therapeutic antibody rituximab. We now report on preclinical studies using this novel system, in which the best transduction protocol, reproducibility, yield, feasibility, and functionality of the transduced T lymphocytes have been investigated with a large donor series. Wild-type human CD20 cDNA was transduced into human T lymphocytes, using a Moloney-derived retroviral vector. Alternative protocols were tested by employing either one or four spinoculations (in which cells are centrifuged in the presence of retroviral vector supernatant) and stimulating T cells with phytohemagglutinin (PHA) or anti-CD3/CD28. One spinoculation alone was sufficient to obtain approximately 30% CD20-positive cells within four experimental days. Four spinoculations significantly increased transduction to 60%. A small difference in transduction efficiency was observed between the two stimulation methods, with PHA being superior to anti-CD3/CD28. Transduced cells could be purified on immunoaffinity columns, with purity reaching 98% and yield being on average 50%. Finally, 86-97% of immunoselected T lymphocytes could be killed in vitro with rituximab and complement. More importantly, the CD20 transgene did not alter the functionality of T lymphocytes with respect to allogeneic recognition and cytotoxic response, anti-Epstein-Barr virus cytotoxic response, antigenic response to tetanus toxoid antigen, interleukin 2 (IL-2), IL-4, and interferon gamma production; chemotaxis in the presence of stromal cell-derived factor 1, phenotype for several activation markers including HLA-DR, CD25, CD69, and CD95, and T cell repertoire.

Genetic modification of human T cells with CD20: a strategy to purify and lyse transduced cells with anti-CD20 antibodies.

A retroviral vector has been constructed that contains the human CD20 cDNA under the control of the Moloney murine leukemia virus (Mo-MuLV) LTR. Freshly isolated mononuclear cells are infected for three consecutive days in the presence of PHA and hrlL-2, and a mean 15.9% of the cells (range, 6.5 to 31.7%) acquire a CD3+CD20+ phenotype. Transduced T lymphocytes grow and expand in vitro for up to 3 weeks like mock-infected cells and, as observed for the T lymphoblastoid CEM cell line, CD20 expression is maintained for several months with no change in the growth curve of the cells. CD20-expressing CEM and fresh T lymphocytes can be positively immunoselected on columns using different anti-CD20 antibodies. Exposure to monoclonal chimeric anti-CD20 IgG1(kappa) Rituximab antibody (Roche), in the presence of complement, results in effective and rapid killing of the transduced CD3+CD20+ human T cells in vitro. This approach represents a new and alternative method to gene manipulation with "suicide" genes for the production of drug-responsive T cell populations, a crucial step for the future management of graft-versus-host disease in bone marrow transplant patients.

C-myb, but not B-myb, upregulates type I collagen gene expression in human fibroblasts.

C-myb and B-myb belong to the myb family of transcription factors. We have shown previously that c-myb is deregulated in fibroblasts from systemic sclerosis (scleroderma) patients relative to normal fibroblasts. Scleroderma fibroblasts are known to express elevated levels of collagen genes and transforming growth factor beta is known to be a pro-fibrotic cytokine and to induce transcription of type I collagen genes. We have therefore investigated the role of c-myb and B-myb in the regulation of type I collagen genes in response to transforming growth factor beta in normal human fibroblasts. We show that, in these cells, transforming growth factor beta treatment induces c-myb as well as collagen alpha1(I) and alpha2(I) gene expression, but not B-myb. Furthermore we demonstrate by cotransfection assays that c-myb can upregulate alpha1(I) and alpha2(I) collagen promoters by 6-10-fold whereas B-myb is inactive. The activity of c-myb on both type I collagen promoters requires a functional c-myb DNA binding domain suggesting a direct interaction between c-myb and these promoters. Indeed c-myb is active also on a 500 bp fragment of the alpha2(I) collagen promoter and can bind to this fragment in electrophoretic mobility shift assays. Finally, we show that anti-c-myb anti-sense treatment reduces alpha1(I) and to a lesser extent alpha2(I) collagen gene expression. These data strongly suggest that c-myb, but not B-myb, plays a direct role in the upregulation of type I collagen gene expression in response to transforming growth factor beta.

The A-myb transcription factor in neoplastic and normal B cells.

The myb family of transcription factors has been strongly implicated in the regulation of cell growth and differentiation in the haematopoietic system. The v-myb oncogene, carried by avian defective retroviruses, causes leukaemias in the chicken and transforms haematopoietic cells in vitro. Its normal cellular equivalent c-myb, has been shown to promote the proliferation and block the differentiation of haematopoietic cells in several experimental models and is required for fetal haematopoiesis. Two other members of the family have been cloned more recently, A-myb and B-myb, which show sequence homology with c-myb in several domains, of which the DNA binding domain as well as other regulatory domains. Both have been shown to be transcription factors. B-myb is also involved in the control of proliferation and differentiation, but, unlike c-myb, it is expressed in many cell types. The third member of the family, A-myb, shows the most restricted pattern of expression, suggesting a very specific role for this transcription factor. A-myb is expressed in a subpopulation of normal B lymphocytes activated in vivo and localised in the germinal center of peripheral lymphoid organs and is not detected at significant levels in all other mature or immature haematopoietic populations studied, including bone marrow cells, T lymphocytes, granulocytes, monocytes, either at rest or after in vitro activation. These studies indicate that A-myb plays a role during a narrow window of normal B cell differentiation. A-myb expression has also been studied in a wide range of neoplastic B cells, representing the whole spectrum of B cell differentiation. A-myb is strongly expressed in Burkitt's lymphomas (BL) and slg+ B-acute lymphoblastic leukaemias (B-ALL) and not in all other leukaemias/lymphomas tested, with the exception of a subset of CLL (about 25% of cases). It is intriguing that the A-myb genome has been localised relatively close to the c-myc gene on chromosome 8, suggesting that the c-myc translocation in BL and B-ALL may affect A-myb transcription. Studies are in progress to investigate the functional relationship between A-myb and c-myc, particularly in the context of BL cells and to determine whether A-myb is deregulated in these cells.

Cytokine Induced Killer (CIK) cells for the treatment of haematological neoplasms.

Cytokine Induced Killer (CIK) cells are in vitro activated human CD8 T cells which have maintained several characteristics of T-EMRA cells and additionally acquired non specific anti tumoral cytotoxicity and CD56 overexpression, thus representing a cell population with double T and NK phenotype. Due to their in vivo intratumoral homing and lack of Graft versus Host (GVH) reactivity, CIK cells have been extensively used in cancer patients either in autologous or allogeneic contexts. Here we summarise CIK main biological features as well as their most prominent clinical results.

The histone deacetylase inhibitor ITF2357 selectively targets cells bearing mutated JAK2(V617F).

We investigated the activity of ITF2357, a novel histone deacetylase inhibitor (HDACi) with antitumor activity, on cells carrying the JAK2(V617F) mutation obtained from polycythemia vera (PV) and essential thrombocythemia (ET) patients as well as the HEL cell line. The clonogenic activity of JAK2(V617F) mutated cells was inhibited by low concentrations of ITF2357 (IC(50) 0.001-0.01 microM), 100- to 250-fold lower than required to inhibit growth of normal or tumor cells lacking this mutation. Under these conditions, ITF2357 allowed a seven fold increase in the outgrowth of unmutated over mutated colonies. By western blotting we showed that in HEL cells, ITF2357 led to the disappearance of total and phosphorylated JAK2(V617F) as well as pSTAT5 and pSTAT3, but it did not affect the wild-type JAK2 or STAT proteins in the control K562 cell line. By real-time PCR, we showed that, upon exposure to ITF2357, JAK2(V617F) mRNA was not modified in granulocytes from PV patients while the expression of the PRV-1 gene, a known target of JAK2, was rapidly downmodulated. Altogether, the data presented suggest that ITF2357 inhibits proliferation of cells bearing the JAK2(V617F) mutation through a specific downmodulation of the JAK2(V617F) protein and inhibition of its downstream signaling.

Gemtuzumab ozogamicin (Mylotarg) has therapeutic activity against CD33 acute lymphoblastic leukaemias in vitro and in vivo.

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated with the cytotoxic drug calicheamicin and approved for the treatment of relapsed acute myeloid leukaemia. As approximately 18% of acute lymphoblastic leukaemias (ALL) are also CD33 positive, we have investigated the cytotoxic activity of GO on CD33+ ALL cells in vitro and in vivo. 10 ng/ml GO induced 30-95% inhibition of thymidine uptake and 30-70% cell death in four freshly isolated and one in vivo passaged CD33+ ALL-cell cultures. Furthermore, an in vivo model of a CD33+ ALL carrying the Philadelphia chromosome [t(9;22)] was established. 5 x 10(6) ALL-2 cells inoculated in the tail vein of severe combined immunodeficient mice engrafted into haematopoietic organs, reaching a mean of 70%, 61% and 69% human CD45+ cells in bone marrow, spleen and liver, respectively, at 35 d. To test the therapeutic activity of GO, 50 or 100 microg immunotoxin was inoculated i.p. on days 7, 11 and 15 following tumour-cell inoculation. GO treatment dramatically inhibited expansion of ALL-2 cells in all tested organs and increased survival of tumour-injected animals by 28-41 d, relative to controls. These data demonstrated that GO is active both in vitro and in vivo against CD33+ ALL cells.

A single point mutation in the v-ets oncogene affects both erythroid and myelomonocytic cell differentiation.

The v-myb, ets-containing avian leukemia virus E26 is unique in its capacity to transform both erythroblasts and myeloblasts. Previous studies showing that v-myb is sufficient for the transformation of myeloid cells failed to definitively establish the role of the v-ets gene. We have now isolated a mutant of E26, ts1.1, that is temperature-sensitive for erythroid cell transformation and that we found to contain a single mutation in the v-ets gene. Surprisingly, myeloid cells transformed by this mutant showed an altered phenotype relative to wild-type-transformed cells, in that they resemble promyelocytes. In addition, infection of mature macrophages with ts1.1 led to their transformation and conversion into promyelocyte-like cells. We conclude that the v-ets domain of the p135gag-myb-ets protein of E26 has an effect on both erythroid and myeloid cell differentiation, suggesting a possible role for the c-ets/c-myb genes in the commitment of hematopoietic cells towards specific lineages.

B cells in patients with X-linked and 'common variable' hypogammaglobulinaemia.

E- cells from three patients with X-linked and three patients with non-familial 'common variable' hypogammaglobulinaemia were stained by indirect immunofluorescence with a panel of B cell specific monoclonal antibodies (anti-Bp95, Bp35 and Bp135). The number of B cells detected with the pan-B cell antibodies was variable between patients. One patient in each group was found to have near normal numbers of circulating B cells although those of the X-linked patient were clearly immature.

The squatting position for the second stage of labor: effects on labor and on maternal and fetal well-being.

A cohort study was designed to assess the effects of maternal squatting position for the second stage of labor on the evolution and progress of labor, and on maternal and fetal well-being. Outcomes from 200 squatting births, randomly selected from a sample of 1000, were compared with 100 semirecumbent births, randomly selected from a sample of 300. Data collection was by chart review. The two groups were similar with respect to most antepartal, intrapartal, and socioeconomic variables likely to affect labor outcomes. The mean length of the second stage of labor was 23 minutes shorter in squatting primiparas and 13 minutes shorter in squatting multiparas than in semirecumbent women. Squatting women required significantly less labor stimulation by oxytocin during second stage (P = 0.0016), and they showed a trend toward fewer mechanically assisted deliveries. Significantly fewer and less severe perineal lacerations occurred, and fewer episiotomies were performed in the squatting group (P = 0.0001). No statistically significant differences were found between groups for third-stage complications and infant complications.

Pathways of human B-lymphocyte activation blocked by B-cell specific monoclonal antibodies.

The data presented in this report demonstrate that antibodies directed against B-cell specific surface antigens can block different modes of B-cell activation. The anti-CD 19 antibody HD37 strongly blocks the stimulation of thymidine incorporation induced by anti-mu and MLR supernatant and also partially inhibits the growth of long-term EBV-transformed cell lines. The anti-CD20 antibody B1, on the other hand, has little effect on anti-mu activation but prevents the stimulation of thymidine incorporation and transformation by EBV. We conclude that different mechanisms operate during B-cell activation by anti-mu and EBV involving different B-cell surface molecules.

Independent regulation of c-myc, B-myb, and c-myb gene expression by inducers and inhibitors of proliferation in human B lymphocytes.

Although a detailed picture is emerging about the nature of the second messengers involved in B cell activation and proliferation, little is yet known about the intracellular events taking place further downstream. The c-myb proto-oncogene, the structurally related B-myb gene, and c-myc probably code for transcription factors, have been demonstrated to be necessary for the proliferation of hemopoietic cells, and their expression is indeed induced after mitogenic stimulation of T and B lymphocytes. They are therefore likely to be key elements in the regulation of gene expression during proliferation. We have set out to study the regulation of the expression of these two myb genes and of that of c-myc in relation to entry into the different phases of the cell cycle during mitogenic stimulation of resting human B lymphocytes. Resting tonsillar B cells stimulated with the anti-CD20 antibody 1F5 alone are induced to enter the G1 but not the S phase of the cell cycle, whereas co-stimulation with the anti-CD40 antibody G28.5 further drives them to enter the S phase and proliferate. The G28.5 antibody alone has been reported to partially activate and increase the alertness of resting B cells without inducing them to enter G1. In this report we show that increasing the strength of the activating signal leads to progressive induction of the proliferation-related genes studied. Thus the G28.5 antibody alone induces c-myc mRNA only in resting B cells, 1F5 induces both c-myc and B-myb, and the full mitogenic signal given by both antibodies together is accompanied by increased expression of all three--c-myc, B-myb, and c-myb genes. In addition, using a semi-quantitative polymerase chain reaction method, we show that different inhibitors of B cell proliferation, namely, cyclosporin A, an anti-CD19 antibody (HD37), and transforming growth factor beta 1 (TGF-beta 1), inhibit differentially the induction of these same genes after mitogenic stimulation of B cells. Whereas cyclosporin A inhibits induction of all three genes, TGF-beta 1 specifically blocks B-myb induction and CD19 has little effect on either of the genes tested. We conclude that c-myb, B-myb, and c-myc are regulated independently from one another, that induction of c-myc and B-myb together is not sufficient to trigger B cell proliferation, and we suggest that expression of all three is a prerequisite for proliferation to occur.